Standard Flow Cytometry Research Articles

Deep immunophenotypic dissection and clinical impact of T cells in the follicular lymphoma microenvironment.

Follicular lymphoma (FL) is an indolent B cell lymphoma with a heterogenous disease course, and patients may not require immediate treatment upon diagnosis. Scrutiny of its microenvironment may provide key insights into lymphomagenesis and enhancement of therapeutic options. We analyzed the T-cell composition of a large, well-annotated follicular hyperplasia (FH; n=43) cohort utilizing standardized high dimensionality flow cytometry (>150,000 cells analyzed/sample) and a novel reproducible analytical pipeline leading to identification of even minor T-cell subsets. This baseline reference set was compared to prospectively collected FL samples (n=91) from untreated patients (FL-UT) and patients with relapsed/refractory disease (FL-RR). Compared to FH, both FL-UT and FL-RR specimens exhibited depletion of CD4+ and CD8+ naive subsets and were characterized by an immune suppressive microenvironment enriched in specific inhibitory T-cells, along with exhausted memory T-cells overexpressing varying combinations of immune checkpoint receptors. FL specimens showed enrichment of T follicular regulatory cells (TFR) and two highly suppressive regulatory T-cell (Treg) populations expressing TIGIT and CTLA4 (TC) and PD1, TIGIT, CTLA4, and TIM3 (PTCTi). FL-UT cases with either increased T-reg TC or increased T follicular helper cells (TFH) showed reduced time to first treatment (.

A high-throughput, microplate reader-based method to monitor in vitro HIV latency reversal in the absence of flow cytometry.

J-Lat cells are derivatives of the Jurkat CD4+ T cell line that contain a non-infectious, inducible HIV provirus with a GFP tag. While these cells have substantially advanced our understanding of HIV latency, their use by many laboratories in low and middle-income countries is restricted by limited access to flow cytometry. To overcome this barrier, we describe a modified J-Lat assay using a standard microplate reader that detects HIV-GFP expression following treatment with latency-reversing agents (LRAs). We show that HIV reactivation by control LRAs like prostratin and romidepsin is readily detected with dose dependence and with significant correlation and sensitivity to standard flow cytometry. For example, 10μM prostratin induced a 20.1±3.3-fold increase in GFP fluorescence in the microplate reader assay, which corresponded to 64.2±5.0% GFP-positive cells detected by flow cytometery. Similarly, 0.3μM prostratin induced a 1.7±1.2-fold increase compared to 8.7±5.7% GFP-positive cells detected. Using this method, we screen 79 epigenetic modifiers and identify CUDC-101, molibresib, and quisinostat as novel LRAs. This microplate reader-based method offers accessibility to researchers in resource-limited regions to work with J-Lat cells and more actively participate in global HIV cure research efforts.

The Role of Th17/Treg Imbalance, FeNO, Eosinophils, IgE and Their Correlation with Lung Function Parameters with Asthma-Chronic Obstructive Pulmonary Disease

This study explored the link between clinical features, immune markers, and asthma-chronic obstructive pulmonary disease overlap (ACO), aiming to enhance diagnostic precision and tailor treatment. The study included 60 patients per group: COPD patients, ACO patients, and healthy controls. Biological indicators such as fractional exhaled nitric oxide (FeNO), eosinophils, immunoglobulin E (IgE), T helper (Th) 17 cell counts, regulatory T-cell (Treg) counts, and cytokine levels of interleukin-17 (IL-17) and interleukin-10 (IL-10) were measured using standard enzyme-linked immunosorbent assay and flow cytometry techniques. Elevated Th17 cells, IL-17, and Th17/Treg ratio, alongside reduced IL-10 and Treg levels, were observed in COPD and ACO patients. ACO patients showed worse lung function, with a negative correlation between FeNO, Th17 cells, Th17/Treg ratio, IL-17, and lung function indices, and a positive correlation with residual volume/total lung capacity (RV/TLC) ratio. The study suggests that Th17/Treg imbalance, FeNO, eosinophils, and IgE could be key in ACO pathogenesis, potentially aiding early diagnosis and targeted treatment. Future research may utilize these findings to develop preventative and therapeutic strategies for ACO.

Comparison of Standard and Point-of-Care CD4+ T Lymphocyte Measurement Methods in HIV-1 Infected Turkish Patients.

Background and Objectives: CD4+ T lymphocytes are the primary targets of HIV infection. CD4+ T lymphocyte count is an indicator of immune competence. In this study, we aimed to compare standard flow cytometry and point-of-care (POC) CD4+ T lymphocyte in terms of cost, effectiveness, reliability, time, and the use of this method for disease. Materials and Methods: This study includes 113 patients. CD4+ T lymphocyte count and percentage were evaluated by flow cytometry and POC. Also, hemoglobin (Hb) level was studied. The data obtained by two methods are compared. Results: When the two methods were compared, intraclass coefficients demonstrated a good consistency for Hb (ICC = 0.849) and CD4+ T lymphocyte percentage (ICC = 0.803). For CD4+ T lymphocyte count, consistency was moderate, ICC = 0.651, but still statistically significant (p < 0.001). Conclusions: In resource-limited countries, virological monitoring with HIV RNA cannot be performed at any time because it is expensive. However, CD4+ T lymphocyte count and percentage monitoring is important in predicting treatment success. POC results are in good consistency with the standard method, and it is also a test that can be used due to being cheap, easy, and quick.

Tamm-Horsfall protein augments neutrophil NETosis during urinary tract infection.

Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THP-deficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared to WT mice. Furthermore, THP-deficient mice displayed impaired urinary NETosis during UTI. To investigate the impact of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways, enhances NETosis in an ROS-dependent manner, and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.

Blood integrin- and cytokine-producing T cells are associated with stage and genetic risk score in age-related macular degeneration

Age-related macular degeneration (AMD) remains a leading cause of vision loss in the geriatric population. There are age-related changes in peripheral blood leukocyte composition, but their significance for AMD remains unclear. We aimed to determine changes in immune cell populations in the blood of AMD patients. A standardized 31-parameter flow cytometry analysis was conducted on peripheral blood mononuclear cells from 59 patients with early and advanced AMD and 39 controls without AMD, all older than 65 years. Fundus photography and optical coherence tomography were used to classify disease stages and a custom genotype array was used to compute an AMD genetic risk score based on 52 AMD disease risk variants (GRS-52). A generalized linear regression model corrected for age, sex, and smoking status revealed that AMD patients showed decreased frequencies of CD4+ T helper cell population expressing Integrin Alpha E (CD103) (Padj = 0.019). We further noted that early AMD was characterized by increased interleukin-4 (IL-4)-producing CD4+ T helper cells (Padj = 0.013; <0.001), as well as IL-4-producing cytotoxic CD8+ T cells (Padj = 0.016; <0.001). Reclassification of samples based on the GRS-52 revealed that IL-17-producing T cells decreased incrementally across GRS-52 categories. In AMD, alterations in peripheral blood leukocyte populations are associated with genetic risk score and disease stage and include specifically IL-4 and IL-17A cytokine-producing and CD103 integrin-expressing T cell populations.

Improved identification of clinically relevant Acute Leukemia subtypes using standardized EuroFlow panels versus non-standardized approach.

Rare acute leukemia (AL) components or subtypes such as blastic plasmacytoid dendritic cell neoplasm (BPDCN) or early T-cell precursor acute Lymphoblastic Leukemia (ETP-ALL) can be difficult to detect by routine flow cytometry due to their immunophenotypes overlapping with other poorly differentiated AL. We hypothesized that using standardized EuroFlow™ Consortium approach could better diagnose such entities among cases that previously classified as acute myeloid leukemia (AML)-M0, AML with minimal differentiation, AML with myelodysplasia-related changes without further lineage differentiation, and AL of ambiguous lineage. In order to confirm this hypothesis and assess whether these AL subtypes such as BPDCN and ETP-ALL had previously gone undetected, we reanalyzed 49 banked cryopreserved sample cases using standardized EuroFlow™ Consortium panels. We also performed target sequencing to capture the mutational commonalities between these AL subtypes. Reanalysis led to revised or refined diagnoses for 23 cases (47%). Of these, five diagnoses were modified, uncovering 3 ETP-ALL and 2 typical BPDCN cases. In 12 AML cases, a variable proportion of immature plasmacytoid dendritic cell and/or monocytic component was newly identified. In one AML case, we have identified a megakaryoblastic differentiation. Finally, in five acute lymphoblastic leukemia (ALL) cases, we were able to more precisely determine the maturation stage. The application of standardized EuroFlow flow cytometry immunophenotyping improves the diagnostic accuracy of ALs and could impact treatment decisions.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

A-220 Standardized flow cytometry for the detection and characterization of circulating CTCL cells: Update on a multicenter study

A-220 Standardized flow cytometry for the detection and characterization of circulating CTCL cells: Update on a multicenter study

Quality Assessment of Cryopreserved Peripheral Blood Stem Cell Products: Evaluation of Two Methods for Flow Cytometric Viability Testing.

The standard flow cytometry method for viability testing using 7-aminoactinomycin D (7-AAD) determines cells in necrosis and late apoptosis. The colony-forming unit (CFU) assay, which evaluates the proliferation ability of HSCs, is also used in graft quality assessment despite known deficiencies that make this assay impractical in routine clinical settings. The aim was to compare the effectiveness of the flow cytometry 7-AAD/annexin V method with the 7-AAD method in assessing the quality of HSCs in autologous and allogeneic peripheral blood stem cell (PBSC) products. Thirty autologous and 30 allogeneic fresh and thawed cryopreserved PBSC products were included in this study. The viability of HSCs was determined using the 7-AAD method and 7-AAD/annexin V method on a flow cytometer, while their clonogenic capacity was assessed by CFU assay. There was an excellent correlation for CD34+ cell viability between the 7-AAD and the 7-AAD/annexin V method for fresh samples (Rs = 0.930, p < 0.001) and a good correlation for thawed PBSC samples (Rs = 0.739, p < 0.001). Excellent correlation was observed for post-thaw CD34+ cell recovery between the two methods for viability (Rs = 0.980, p < 0.001). Statistical analysis showed a weak correlation between CFU-GM recovery and CD34+ cell recovery, regardless of which viability testing method was used (7-AAD method p = 0.021, Rs = 0.298; 7-AAD/annexin V method p = 0.029, Rs = 0.282). Results of this study showed that in the quality assessment of cryopreserved PBSC product viability, the 7-AAD/annexin V method had no added value compared to the 7-AAD method, which was suitable enough for routine quality control of cryopreserved autologous and allogeneic PBSC samples.

Surface Immune Checkpoints as Potential Biomarkers in Physiological Pregnancy and Recurrent Pregnancy Loss.

Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11-14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression.

Cellular Effects of Enterobacteriaceae Polysaccharide Colanic Acid.

Colanic acid (CA) is an exopolysaccharide found in Enterobacteriaceae. Recently, its ability to stimulate physical activity in mice and to prolong the lifespan of invertebrates has been described. In the current work, we use standard MTT assay, fluorescence microscopy, and flow cytometry to describe CA action on several cell lines of different origins. We observed slight antiproliferative activity against colorectal cancer (HCT-116), neuroblastoma (IMR-32), and myoblast (C2C12) cell lines at a concentration of 256 μg/mL, while other cell lines of non-cancerous origin (Vero, HPF) did not show any decrease in the MTT assay. In all cell lines, we observed a rearrangement of mitochondria localization using fluorescence microscopy. CA induces cell differentiation in the myoblast cell line (C2C12) at concentrations of 50-200 μg/mL. Briefly, we observed that the number of apoptotic cells increased and the metabolic activity in the MTT assay decreased, which was accompanied by changes in cell morphology, the quantity of ROS, and the potential of the mitochondrial membrane. Taken together, these results indicate that CA is specific in cytotoxicity to cell lines of different origins and can impact mitochondria and differentiation, consistent with its potential geroprotective function.

Flow cytometry in the differential diagnosis of myelodysplastic neoplasm with low blasts and cytopenia of other causes.

Myelodysplastic neoplasms (MDS) are characterized by cytopenia, morphologic dysplasia, and genetic abnormalities. Multiparameter flow cytometry (FCM) is recommended in the diagnostic work-up of suspected MDS, but alone is not sufficient to establish the diagnosis. Our aim was to investigate the diagnostic power of FCM in a heterogeneous population of patients with cytopenia, excluding cases with increased blast count. We analyzed bone marrow samples from 179 patients with cytopenia (58 MDS, 121 non-MDS) using a standardized 8-color FCM method. We evaluated the sensitivity, specificity, and accuracy of several simple diagnostic approaches, including Ogata score, extended Ogata score, the WHO and ELN iMDSFlow recommended "3 aberrations in two cell compartments method," and the combination of the Ogata score and "3 aberrations in two cell compartments method." The patients were followed until the diagnosis was confirmed, with a median follow-up of 2months (range 0.2-27). The combination of Ogata score and "3 aberrations in two cell compartments method" achieved the highest diagnostic accuracy (78%) with sensitivity and specificity 61% and 86%, respectively. When using only the "3 aberrations in two cell compartments method," the accuracy was 77% with a sensitivity of 72% and a specificity of 79%. The most frequently observed etiologies among the false positive cases were substrate deficiencies, inflammation/infection, or toxic effects. MDS can be excluded in all these cases after a thorough clinical evaluation and a relatively short follow-up. FCM remains an important but supplementary part in an integrated diagnostic process of MDS with low blasts. The combination of the Ogata score and the "3 aberrations in two cell compartments method" slightly improves accuracy compared to the detection of "3 aberrations in two cell compartments method" alone.

Unveiling Paclitaxel-Induced Mesenchymal Stem Cells: orchestrating Nrf2 Modulation and Apoptosis in CD44+/CD24- Cancer Stem Cells.

Mesenchymal Stem Cells (MSCs) and Cancer Stem Cells (CSC) play pivotal roles in cancer progression and therapeutic responses. This study aimed to explored the effect of MSCs induced by paclitaxel on CSC expressing the CD44+/CD24- phenotype, focusing on Nrf2 modulation and apoptosis induction. MSCs were characterized for adherence, differentiation potential, and surface markers via standard culture, staining assays, and flow cytometry, respectively. CSCs isolated from MDA-MB-231 using MACS and were characterized based on morphology and CD44+/CD24- expression. Co-culture experiments evaluated the cytotoxic effect of Paclitaxel-induced MSCs on CSC viability using MTT assays. Flow cytometry analysis assessed apoptosis induction via annexin V-PI staining and Nrf2 and Caspase-3 gene expression were measure by qRT-PCR analysis. MSCs exhibited typical adherence and differentiation capabilities, confirming their mesenchymal lineage. CSCs displayed an elongated morphology and expressed CD44+/CD24-, characteristic of stem-like behavior. Paclitaxel induced dose-dependent Nrf2 gene expression in MSCs. Co-culture with Paclitaxel-induced MSCs reduced CSC viability in a dose-dependent manner, with a significant decrease observed at a 5:1 MSCs:CSC ratio. Co-culture decreased the Nrf2 gene expression and increased apoptosis in CSCs, with higher caspase-3 gene expression compared to solitary paclitaxel treatment. Paclitaxel-induced MSCs decreased Nrf2 expression and significantly decreased CSC viability while enhancing apoptosis. This suggests a potential strategy to mitigate paclitaxel resistance in CD44+/CD24- CSCs. Leveraging Paclitaxel-induced MSCs presents a promising avenue for targeting Nrf2 and promoting apoptosis in CSCs, potentially improving the efficacy of chemotherapy and addressing resistance mechanisms in cancer treatment.

A potential best-in-class BCMA-CD19 bispecific CART with advanced safety by self-inhibiting IFNG signaling during CRS.

2502 Background: CART cells have demonstrated remarkable clinical efficacy in treating hematological cancers. However, CRS remains as a major challenge in clinical application, with multiple cytokines significantly elevated. IL6 signaling blockade by Tocilizumab has become a standard treatment to relieve CRS in patients. Our previous studies suggested that CAR T secreting IL6 antagonist could effectively maintain IL6 at very low levels during CRS. Furthermore, we observed that a huge quantity of tumor cells in one patient was significantly reduced after CART infusion, but a low level of IFNG with only grade 2 CRS was present. Therefore, we hypothesized that high level of IFNG might not be necessary to CART clinical efficacy and direct blockade of IFNG signaling by CART secreting an IFNG antagonist (designated as SAFET) serves an interesting approach to effectively reduce CRS toxicity. Although IFNG is usually thought important in T cell cytotoxicity, our results indicated that IFNG KO or autonomous secretion of IFNG antagonist did not affect the killing activity of CART cells. Methods: This pilot phase 1 study of SAFET BCMA/CD19 BiCART self-inhibiting IFNG signaling is a single arm study conducted in China, enrolling refractory/relapsed multiple myeloma patients. The patients had received more than 3 lines of prior therapies including at least a proteasome inhibitor and an immunomodulatory agent and were refractory to the last line of treatment. Lymphodepletion was performed with fludarabine and cyclophosphamide prior to the CART infusion. Following 2-14 days of rest, patients received a single infusion of SAFET BCMA/CD19 BiCART, at the dose of 0.4-1.0 × 108 CAR+ T cells/patient. The primary objectives of this study were to evaluate the safety and efficacy of SAFET BCMA/CD19 BiCART. The pharmacokinetics of SAFET BCMA/CD19 BiCART was investigated by quantitative PCR based detection of CAR vector copies in peripheral blood. Minimal residual disease (MRD) negativity was assessed by standardized multicolor flow cytometry analysis of bone marrow aspirate. Results: 10/11 R/R MM patients achieved CR and 1 achieved VGPR. Among the 10 CR patients, 7 remained CR after treatment with a median PFS 858 days; 1 patient showed relapse at Days 215; 2 patients showed MRD positive relapse at Days 637 and 733, and then received KRd and/or Kd treatment to achieve SD and MRD negative CR. Interestingly, the remarkable short and long term remission suggested that CART self-inhibiting IFNG signaling did not impair the CART clinical efficacy. Notably, minimal CRS was observed, including 6 grade 0, 2 grade 1, 2 grade 2, and 1 grade 3 who displayed mild transient hypotension for only 1 day while there were 79.5% blast tumor cells in the bone marrow before CART therapy. Conclusions: These results suggest BCMA-CD19 bispecific CART self-inhibiting IFNG signaling is promising in translation to a best-in-class treatment for MM. Clinical trial information: ChiCTR2000032124.

Towards a Point-of-Care Test of CD4+ T Lymphocyte Concentrations for Immune Status Monitoring with Magnetic Flow Cytometry.

For the treatment of human immunodeficiency virus (HIV)-infected patients, the regular assessment of the immune status is indispensable. The quantification of CD4+ T lymphocytes in blood by gold standard optical flow cytometry is not point-of-care testing (POCT) compatible. This incompatibility is due to unavoidable pre-analytics, expensive and bulky optics with limited portability, and complex workflow integration. Here, we propose a non-optical, magnetic flow cytometry (MFC) workflow that offers effortless integration opportunities, including minimal user interaction, integrated sample preparation and up-concentration, and miniaturization. Furthermore, we demonstrate immunomagnetic CD4+ T lymphocyte labeling in whole blood with subsequent quantification using sheath-less MFC. Showing linearity over two log scales and being largely unimpaired by hematocrit, evidence is provided for POCT capabilities of HIV patients.

A 3-step method for preparing cryopreserved samples of apheresis products for post-thaw analysis yields a higher percentage of viable cells.

Standard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab-induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities. We developed a novel method whereby thawed samples were diluted step-wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution. Twenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter. Slow, step-wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

A novel flow cytometry panel to identify prognostic markers for steroid-sensitive forms of idiopathic nephrotic syndrome in childhood.

The clinical evolution of steroid-sensitive forms of pediatric idiopathic nephrotic syndrome (INS) is highly heterogeneous following the standard treatment with prednisone. To date, no prognostic marker has been identified to predict the severity of the disease course starting from the first episode. In this monocentric prospective cohort study we set up a reproducible and standardized flow cytometry panel using two sample tubes (one for B-cell and one for T-cell subsets) to extensively characterized the lymphocyte repertoire of INS pediatric patients. A total of 44 children with INS at disease onset were enrolled, sampled before and 3 months after standard induction therapy with prednisone and followed for 12 months to correctly classify their disease based on relapses. Age-matched controls with non immune-mediated renal diseases or with urological disorders were also enrolled. Demographical, clinical, laboratory and immunosuppressive treatment data were registered. We found that children with INS at disease onset had significantly higher circulating levels of total CD19+ and specific B-cell subsets (transitional, mature-naïve, plasmablasts/plasmacells, CD19+CD27+, unswitched, switched and atypical memory B cells) and reduced circulating levels of Tregs, when compared to age-matched controls. Prednisone therapy restored most B- and T-cell alterations. When patients were subdivided based on disease relapse, relapsing patients had significantly more transitional, CD19+CD27+ memory and in particular unswitched memory B cells at disease onset, which were predictive of a higher risk of relapse in steroid-sensitive patients by logistic regression analysis, irrespective of age. In accordance, B-cell dysregulations resulted mainly associated with steroid-dependence when patients were stratified in different disease severity forms. Of note, Treg levels were reduced independently from the disease subgroup and were not completely normalized by prednisone treatment. We have set up a novel, reproducible, disease-specific flow cytometry panel that allows a comprehensive characterization of circulating lymphocytes. We found that, at disease onset, relapsing patients had significantly more transitional, CD19+CD27+ memory and unswitched memory B cells and those who are at higher risk of relapse had increased circulating levels of unswitched memory B cells, independently of age. This approach can allow prediction of clinical evolution, monitoring of immunosuppression and tailored treatment in different forms of INS.

High Dimensional Clustering and Identification of Immune Cell Hybrid Populations in B-ALL across the Disease Continuum

Introduction: Nearly half of adults with acute lymphoblastic leukemia (ALL) will experience relapse of their disease, and those with relapsed ALL carry a worse prognosis than those who remain in remission. The influence of the tumor immune microenvironment (TME) on relapse and survival outcomes in ALL is not well understood. Mass cytometry (CyTOF) enables immune profiling based on single cell protein expression and can identify phenotypic diversity of cells in the TME that cannot reliably be detected by standard flow cytometry. Recently, T-cell/monocyte hybrid cells have been discovered in patients with uveal melanoma and latent tuberculosis infections. However, cells expressing CD19, a surface antigen expressed on B-ALL cells, and markers of other cells in the TME including T cells and tumor-associated macrophages (TAMs), have not been described in ALL or other hematologic malignancies. The presence of CD3/CD163 hybrid cells in the TME may have predictive and prognostic implications for adults with ALL, as TAMs play a role in suppressing antitumor immunity which can lead to T-cell exhaustion. In this study, we identify the presence of multilineage hybrid cells in peripheral blood (PB) and bone marrow (BM) samples of patients with B-ALL alongside accompanying evolution of the TME at each disease stage. Methods: PB (N=16) and BM aspirate (N=16) isolates were collected from patients with B-ALL at diagnosis, relapse, or remission, as well as from healthy donors (HDs, N=7) for comparison of cellular profiles. A 28-marker CYTOF panel was designed to evaluate cell surface and intracellular markers of interest in the myeloid compartment. Antibodies were purchased pre-conjugated or conjugated in house based on manufacturer guidelines (Standard BioTools). Samples were barcoded with palladium isotopes prior to staining to reduce the impact of batch effect, and were then analyzed on a first generation CyTOF system (Standard BioTools). Following background attenuation and singlet gating in FlowJo (BD), the CATALYST R package enabled deconvolution and visualization of single cell data, including dimensional reduction techniques (tSNE) and clustering of all cell populations. An iterative approach was taken to determine a stable configuration of clusters across sample populations to protect from over-fragmentation of data, further informed by K-means clustering using the elbow method. Heatmaps were then generated based on median scaled expression of markers contained in each cluster. Results: The CyTOF panel identified several different myeloid cell populations in newly diagnosed, relapsed, and remission samples in both PB and BM compared to HDs. Most interestingly, quantifiable levels of a cluster of cells co-expressing CD19, CD3 and CD163 as well as a second cluster of cells expressing CD19 and CD163 were detected in newly diagnosed and relapsed patients, but not in HDs. A single patient with the largest proportion of these cell populations in their PB at the time of relapse presented with an aggressive leukemia variant and relapsed multiple times prior to succumbing to illness. On investigation of BM aspirates, the triple positive hybrid population was found in a similar distribution amongst the newly diagnosed and relapsed patients, as well as affecting one patient who achieved remission. This patient also had a highly aggressive leukemia that was refractory to chemotherapy and immunotherapy, and only achieved this remission following CAR-T cell therapy. She subsequently underwent stem cell transplant and is in remission. Conclusions: Our findings add to the literature highlighting the utility of high parameter single cell capture techniques in detecting rare cell populations in the leukemic TME. Further, we identified two unique hybrid cell populations that are reliably detected across different disease stages in patients with B-ALL. Importantly, these populations persisted following stringent gating protocols to rule out doublets and debris. Although our sample size was too small to demonstrate a prognostic impact of these hybrid cell populations in patients with B-ALL, the identification of these populations alone is a significant finding. Further investigation and patient sample accrual are warranted to better understand the role of these cell types in tumor biology, which may pave the way towards new prognostic models that can leverage well-established immune monitoring techniques.

Single Cell Multi-Omic Analysis of Neoplastic Plasma Cells across the Disease Spectrum Identifies Novel Pathobiologic Mediators and Potential Therapeutic Targets in Multiple Myeloma (MM)

Background: A challenge to developing a full understanding of MM pathobiology using bulk profiling techniques is the large degree of intra-tumoral heterogeneity from minor subclones that, despite their low representation, nonetheless contribute to therapy resistance and disease progression. Moreover, our current therapies tend to be targeted against pathways common to both neoplastic and normal plasma cells (PCs), indicating a need to define novel tumor biology modifiers that could potentially serve to improve risk stratification and as therapeutic targets. Methods: To overcome these limitations, we simultaneously evaluated individual PC single-cell transcriptome and B-cell receptor variable (VDJ) sequences from freshly enriched bone marrow biopsies. These included 33 patients with precursor conditions, 17 each with newly diagnosed symptomatic or relapsed/refractory disease (RRMM), and 3 controls using the 10x Genomics platform. A total of ~320,000 PCs were analyzed, yielding median per sample cell counts of 4,626 and ~300 million total reads. Identical immunoglobulin VDJ sequences defined monoclonal PCs, while diverse VDJ sequences at low frequencies defined polyclonal cells, allowing each patient to serve as their own control. Results: The percentage of monoclonal PCs in active MM was &amp;gt;84%, while precursor stages were more variable (40-100% in smoldering MM; 0.3-82% in monoclonal gammopathy of undetermined significance (MGUS)). Though we could not identify monoclonal PCs in two MGUS cases, we identified them by scRNA-seq in four MGUS patients whose bone marrow showed no clonal cells by standard clinical multiparametric flow cytometry. In differential gene expression analysis where samples were grouped based on the diagnosis, 501 dysregulated genes were identified in monoclonal PCs from symptomatic MM compared to polyclonal PCs. These included genes previously identified as disease modifiers, including Hepatocyte growth factor, Lactate dehydrogenase A, Dickkopf WNT signaling pathway inhibitor 1, CD27, and Lysosomal associated membrane protein family member 5, providing validation for our approach. Next, we identified 279 genes that were commonly dysregulated in monoclonal PCs from symptomatic vs. asymptomatic MM. We also performed unsupervised hierarchical clustering of the top 1000 most variable genes and defined three diagnostic categories. Significant ontology terms characterizing these categories broadly involved antigen processing and recognition in Group 1 (enriched in precursor stages); transcription and translation control in Group 2 (enriched in active MM); and cell division and DNA metabolism in Group 3 (enriched with RRMM) ( A). We focused on dysregulated targets that: showed differential expression in monoclonal vs. polyclonal PCs; expression in most patients within a diagnostic category; a correlation with outcomes in the CoMMpassstudy; and novelty in MM. Among these were Mitotic arrest deficient 2 like 1 (MAD2L1), a component of the mitotic spindle assembly checkpoint, and Methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of S-adenosylmethionine, a key methyl donor in multiple cellular processes, from methionine and ATP. Notably, MAD2L1 inhibition with M2I1, and MAT2A targeting with the allosteric inhibitor AG-270, both produced a dose- and time-dependent inhibition in cell proliferation and reduction in myeloma cell line viability. Importantly, the effects of AG-270 ( B) were associated with induction of markers of apoptosis, including Caspase cleavage, and a reduction of Histone H3 dimethylation. Moreover, they occurred at clinically relevant concentrations based on the known pharmacokinetics of this drug in Phase I testing. Conclusions: Our combined single-cell transcriptomics and VDJ sequencing allowed a greater understanding of heterogeneity among neoplastic PCs compared to each patient's own polyclonal PCs. These analyses can identify putative novel mediators of disease pathobiology that impact prognosis, and that can serve as potential new therapeutic targets using either novel agents, or drug repurposed from other therapeutic areas, setting the stage for their translation to the clinic.